Bacteriophages have been used in strategies for detecting molecules-of-interest. For example, a method employing the bacteriophage M13 has been used to assay for various proteins of interest. In this method, M13 phage displaying peptides fused to pIII, a minor M13 coat protein, have been used to screen for protein binding molecules and antibodies (Scott et al. (1990) Science 249:386; Devlin et al. (1990) Science 249:404). Special M13-derived systems have been used to express antibodies as fusion proteins on the surface of the phage, and techniques have been developed to enrich the population for phage expressing antibodies with desired affinities for an antigen (Garrard et al. (1991) Bio/Technol. 9:1373; Barbas et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:7978). However, the use of M13 in assay methods is limited because M13 infection is not immediately ascertainable. This is because infection by M13 does not provide the cell with compounds required for growth and is not lytic.
Like M13, T4 has been used in assays for various proteins such as nerve growth factor (NGF) (Oger et al. (1974) Proc. Natl. Acad. Sci. (USA) 71:1554-1558). In this assay, T4 was chemically coupled to NGF using glutaraldehyde. The phage was then rendered non-infective by treatment with antibodies against NGF. When unbound NGF was added to the medium, NGF-linked phage was displaced from the antibody and became free to infect Escherichia coli (E. coli). Bacteriophage T4 has also been used to detect antibodies against a wide range of compounds. For example, Becker et al. (Immunochem. (1970) 7:741) used a T4 bacteriophage to detect antibodies against p-azobenzenearsonate. Hurwitz et al. (Eur. J. Biochem. (1970) 7:273) used a T4 bacteriophage to detect and estimate levels of angiotensin-II-beta-amide and its antibodies. Gurari et al. (Eur. J. Biochem. (1972) 26:247) used bacteriophage T4 in the detection of antibodies to nucleic acids. These detection methods involve the chemical modification of the T4 phage resulting in the non-specific exposure on the phage surface of a compound to which the antibodies to be assayed are targeted. Such antibodies render the bacteriophage non-infective, thus enabling the decrease in plaque formation to be used as a measure of the level of antibody present. The T4 system has also been used to measure hapten concentrations (see, e.g., Hurwitz et al. (1970) Eur. J. Biochem. 17:273-277) In this system, T4 is chemically modified such that it exposes the desired hapten non-specifically on its surface. The addition of anti-hapten antibody destroys the infectivity of the phage. Infectivity is restored in the presence of hapten.
Although both the M13 and T4 phage systems can be used to detect the presence of a compound by their ability to become infectious in the presence of that compound, infection by M13 is normally not immediately ascertainable, and T4 infection is lethal. Thus, these systems cannot be used where a quick screening or selection method based on the survival of the infected bacterial cell is desired, such as where a particular cell type is being selected, or when the object of phage infection is to restore the ability of an auxotrophic bacterial cell to survive on its own under a given set of growth conditions. Special M13-derived phagemid systems carry genes which could endow an infected cell with a selective growth advantage (Barbas et al. (1991) Proc. Natl. Acad Sci. (USA) 88:7978). However, these systems have not been used to detect a molecule-of-interest or cells producing such compounds. Furthermore, because gpIII, the M13 protein to which the target molecules are fused, accumulates on the inner membrane facing the periplasm, there are limitations on the nature of the protein fusion. Fusions that are not able to cross the membrane will not be assembled into M13. In addition, in all M13 systems where fusion proteins have been used to display proteins on the outer surface, the displayed protein (or peptide) itself has been the molecule-of-interest.
Thus, what is needed are methods for assaying for molecules-of-interest and for cells producing such molecules which are efficient, accurate, and fast. What are also needed are assay methods which do not have to result in bacterial cell death. Additionally, assay methods utilizing bacteriophage infection are needed for non-proteinaceous molecules of interest and for cells which continuously produce these molecules-of-interest.